Quantitative assessment of prion infectivity in tissues and body fluids by real-time quaking-induced conversion.

Abstract

Prions are amyloid-forming proteins that cause transmissible spongiform encephalopathies through a process involving the templated conversion of the normal cellular prion protein (PrP(C)) to a pathogenic misfolded conformation. Templated conversion has been modelled in several in vitro assays, including serial protein misfolding amplification, amyloid seeding and real-time quaking-induced conversion (RT-QuIC). As RT-QuIC measures formation of amyloid fibrils in real-time, it can be used to estimate the rate of seeded conversion. Here, we used samples from deer infected with chronic wasting disease (CWD) in RT-QuIC to show that serial dilution of prion seed was linearly related to the rate of amyloid formation over a range of 10(-3) to 10(-8) µg. We then used an amyloid formation rate standard curve derived from a bioassayed reference sample (CWD+ brain homogenate) to estimate the prion seed concentration and infectivity in tissues, body fluids and excreta. Using these methods, we estimated that urine and saliva from CWD-infected deer both contained 1-5 LD50 per 10 ml. Thus, over the 1-2 year course of an infection, a substantial environmental reservoir of CWD prion contamination accumulates.